Substance WF16616, process for production thereof, and use thereof

ABSTRACT

1. WF16616 substance represented by the following structural formula (1) or its pharmaceutically acceptable salt. ##STR1## WF16616 substance exhibits an antimicrobial activity, especially an excellent antifungal activity.

FIELD OF THE INVENTION

The present invention relates to WF16616 substance, a process forproducing the same and use thereof. WF16616 substance is a novelsubstance which is isolated from a culture broth of microorganisms,especially imperfect fungi. This substance exhibits an excellentphysiological activity, and is effective as an antimicrobial agent forhumans and other animals, especially as an agent for preventing ortreating diseases caused by microorganisms, molds, other fungi orprotozoans, such as an antifungal agent and an agent for Pneumocystiscarinii pneumonia.

PRIOR ART

A variety of antimicrobial substances have been developed so far, butfew of them are satisfactory with respect to effectiveness,antimicrobial spectrum, safety, production cost and the like. Therefore,the development of novel substances has been in high demand in the art.

Problems to be Solved by the Invention

The present invention has been conducted to meet such a demand in theart and to develop a novel substance that exhibits an outstandingphysiological activity.

Means Taken for Solving the Problems

The present inventors have conducted various investigations to achievethe above-mentioned purpose. Consequently, they have focussed theirattention on fermentative products of microorganisms, and have earnestlyscreened various microorganisms. As a result, they have newly found thatan extract obtained from a culture broth of No. 16616 strain which wasnewly separated from a soil sample collected in Takato-cho, Nagano-kenexhibits an excellent antimicrobial activity, especially an antifungalactivity. They have further studied the physico-chemical properties ofthis substance in detail, and have identified that this substance is anovel substance which has been so far unknown. They have named thissubstance "WF16616". Upon further investigations, they have establishedan industrial process for producing the same, and have completed thepresent invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chart showing a ¹ H nuclear magnetic resonance spectrum of asalt (which is considered to be a sodium salt, a potassium salt, anammonium salt or a mixture thereof) of WF16616 substance.

FIG. 2 is a chart showing a ¹³ C nuclear magnetic resonance spectrum ofa salt (which is considered to be a sodium salt, a potassium salt, anammonium salt or a mixture thereof) of WF16616 substance.

WF16616 substance of the present invention has been found to berepresented by the following structural formula (1) upon studies onphysico-chemical properties and the like which will be later described.##STR2##

WF16616 substance of the present invention is produced by, for example,No. 16616 strain which is a filamentous fungus separated from a soilsample collected in Takato-cho, Kamiina-gun, Nagano-ken. Thismicroorganism is restrictedly grown on various media and forms ayellowish white colony. Further, on a variety of media No. 16616 strainforms an anamorph consisting of a conidiophore having a verticillatephialide and a sticky conidial mass. The phialide consists of a swollenbase and an elongated stem. In view of these morphologicalcharacteristics, the microorganism is identied to belong to the genusTolypocladium W. Gams (Gams, W: Persoonia, 6:185-191, 1971) which is animperfect fungus. The mycological properties thereof are describedbelow.

The cultural properties of this microorganism in a variety of agar mediaare summed up in Tables 1 and 2 below. It was restrictedly grown throughincubation on a potato dextrose agar medium, and was spread in adiameter of from 1.5 to 2.0 cm after incubation at 25° C. for 2 weeks.The colony surface was felty or cottony, and somewhat raised. Thecentral portion and the peripheral portion of the colony were white toyellowish white, and the portion therebetween was grayish brown. Thereverse thereof was olive brown and yellowish white. The conidialstructures were abundantly observed. The colony was restrictedly grownon a corn meal agar medium also, and was spread in a diameter of from1.5 to 2.0 cm under the same conditions. The colony surface was flat,thin and felty. The central portion of the colony was olive gray, andthe peripheral portion was yellowish white. The reverse thereof had thesame color as the surface. The conidial structures were abundantlyobserved.

                  TABLE 1                                                         ______________________________________                                        (1) Cultural properties of No. 16616 strain                                   Medium Cultural properties                                                    ______________________________________                                        Malt extract agar*                                                            Growth:                                                                              restricted, diameter 1.5-2.0 cm.                                       Surface:                                                                             circular, flat, felty, abundant formation of an anamorph, a                   central portion and a peripheral portion are white to light                   yellow (5A3), a portion therebetween is olive brown (4F4).             Reverse:                                                                             a central portion is olive (3F3), a peripheral portion is orange              white (5A2).                                                           Potato dextrose agar                                                          (Difco 0013)                                                                  Growth:                                                                              restricted, diameter 1.5-2.0 cm.                                       Surface:                                                                             circular, felty or cottony, somewhat raised, abundant forma-                  tion of an anamorph, a central portion and a peripheral portion               are white to yellowish white (4A3) a portion therebetween is                  grayish brown (5F3).                                                   Reverse:                                                                             a central portion is olive brown (4F3), a peripheral portion is               yellowish white (4A2).                                                 Czapek's agar*                                                                Growth:                                                                              restricted, diameter 2.0-2.5 cm.                                       Surface:                                                                             circular, cottony, abundant formation of an anamorph, white.           Reverse:                                                                             white to yellowish white (3A2).                                        Sabouraud's dextrose agar                                                     (Difco 0190)                                                                  Growth:                                                                              restricted, diameter 1.0-2.0 cm.                                       Surface:                                                                             circular, cottony, raised, abundant formation of an anamorph,                 a central portion is grayish brown (7E3), a peripheral portion                is yellowish gray (4B2) to light orange (5A3) forming a light                 orange soluble pigment.                                                Reverse:                                                                             olive brown (4F4) or light orange (5A3).                               ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        (2) Cultural properties of No. 16616 strain                                   Medium Cultural properties                                                    ______________________________________                                        YpSs agar                                                                     (Difco 0739)                                                                  Growth:                                                                              restricted, diameter 2.0-2.5 cm.                                       Surface:                                                                             circular flat, felty or cottony, abundant formation of an                     anamorph, white to light orange (4A3).                                 Reverse:                                                                             light orange (4A3).                                                    Corn meal agar                                                                (Difco 0386)                                                                  Growth:                                                                              restricted, diameter 1.5-2.0 cm.                                       Surface:                                                                             circular, flat, thin, felty, abundant formation of an anamorph,               a central portion is olive gray (2F3), a peripheral portion is                yellowish white (3A2).                                                 Reverse:                                                                             a central portion is olive grayish brown (2F2), a peripheral                  portion is yellowish white (3A2).                                      MY20 agar*                                                                    Growth:                                                                              very restricted, diameter 0.5-1.0 cm.                                  Surface:                                                                             circular, raised, felty, abundant formation of an anamorph,                   white to yellowish gray (4B2).                                         Reverse:                                                                             a central portion is yellowish brown (5FA), a peripheral                      portion is bright orange (5A4).                                        ______________________________________                                    

In the above-mentioned cultural properties, the compositions of theasterisked malt extract agar, Czapek's agar and MY20 agar were based onthe JCM catalogue (Nakase T., 5th ed., p. 503, Japan Collection ofMicroorganisms and Life Science Research Information Section of theInstitute of Physical and Chemical Research, Saitama, 1992).

These properties were observed after incubation at 25° C. for 14 daysfrom the inoculation. The colors were described according to MethuenHandbook of Colour (Kornerup, A. and J. H. Wanscher, 3rd ed., p. 525,Methuen, London, 1978).

The morphological properties were determined on the basis of theincubation on a Miura medium (Miura, K. and Kudo, M., Trans. Mycol. Soc.Japan, 11: 116-118, 1970). The conidiophore is macronematous orsemi-macronematous, solitary, colorless to brown and smooth-surfaced; itis directly raised from the surface of the medium or differentiated froman aerial hypha as a short branch. The conidiophore is simple orslightly branched, and forms phialides solitarily or verticillately. Thephialide is colorless and smooth-surfaced, and has a flask-like shapeconsisting of an elliptically swollen base and an elongated cylindricalstem. The base has a size of 5-8(-10)2×-3 μm, and the stem has a size of2.5-5(-6)×1-1.5 μm. Conidia which are produced continuously form asticky mass, and sometimes form a chain. The conidium is colorless,smooth-surfaced, unicellular and globose or subglobose, and has a sizeof 3-4×2.5-3 μm. A vegetative mycelium is smooth-surfaced, septate, andis branched with a color of from dark brown to light brown. The hyphalcell is cylindrical and has a width of from 1.0 to 3.0 μm. Nochlamydospore is observed, but a single phialo-conidium is sometimesformed on the tip of a phialide.

No. 16616 strain can be grown at from 3° to 30° C., and the optimumgrowth temperature is between 16° and 22° C. These data were determinedon a potato dextrose agar (made by Nissui).

The above-mentioned mycological properties were studied. As a result,according to the classification of microorganisms belonging to the genusTolypocladium (Bissett, J.: Can, J, Bot., 61: 1311-1329, 1983), No.16616 strain is similar to Tolypocladium parasiticum Barron 1980. Thismicroorganism was first described by Barron as a parasite of a rotiferthat lives in a soil on the waterside. However, it can also be grownunder pure culture conditions. No. 16616 strain and T. parasiticum aredifferent only with respect to forming of chlamydospore. T. parasiticumformed the chlamydospore both on the culture medium and on the rotifer.Meanwhile, No. 16616 strain only formed a sole phialo-conidium similarto chlamydospore on the tip of a phialide. With respect to the otherproperties, No. 16616 strain agreed with T. parasiticum. Accordingly,No. 16616 strain was identified as a strain of T. parasiticum. This No.16616 strain has been deposited at the National Institute of Bioscienceand Human-Technology of the Agency of Industrial Science and Technologyunder Deposit No. FERM BP-5553 (original deposit date: May 25, 1995).

It should be understood that the specific microorganism which ismentioned only for explanation of the production of WF16616 substancedescribed in the present specification is not critical. The presentinvention includes the use of all of WF16616 substance-productivemutants including synthetic mutants which can be obtained by themutation of the microorganism described in the present invention throughirradiation with X-rays or ultraviolet light or upon use ofN-methyl-N'-nitro-N-nitrosoguanidine or 2-aminopurine and naturalmutants.

WF16616 substance of the present invention can be produced bycultivation (for example, shaking culture or aeration stirring culture)of a WF16616 substance-productive fungus (for example, No. 16616 strain)belonging to filamentous fungi in a nutrient medium containing carbonand nitrogen sources which can be assimilated by this fungus.

Preferable examples of the carbon source include glucose, sucrose,starch, modified starch, fructose, glycerin and other carbohydrates.

Preferable examples of the nitrogen source include oatmeal, yeastextract, peptone, gluten meal, cottonseed meal, cottonseed oil cake,soybean meal, corn steep liquor, dry yeast, wheat germs, peanut flourand chicken bone and meat meal. Also available are inorganic or organicnitrogen compounds such as ammonium salts (for example, ammoniumnitrate, ammonium sulfate ammonium phosphate), urea and amino acids.

These carbon and nitrogen sources are preferably used in combination.However, pure sources are not altogether necessary. Impure sources oftencontain growth factors or micronutrients, and these are sometimesadvantageous.

For example, an inorganic salt may be added to a medium as required.Examples of the inorganic salt include sodium carbonate, potassiumcarbonate, sodium phosphate, potassium phosphate, sodium chloride,potassium chloride, sodium iodide, potassium iodide, magnesium salts,copper salts and cobalt salts.

Especially, when a medium is strongly foamed, liquid paraffin, animaloil, vegetable oil, mineral oil or silicone may be added thereto asrequired.

When the desired substance is mass-produced, aeration stirring cultureis preferable as in the production of other fermentative substances.When the desired substance is produced in a small amount, shakingculture in a flask is preferable.

When the cultivation is conducted in a large tank, it is preferable, forpreventing delaying in the production of WF16616 substance, that aWF16616 substance-productive fungus be first inoculated and incubated ina relatively small amount of a medium and the resulting culture be thenmoved to a large tank where it is cultivated for the production of thesubstance. In this case, the compositon of the medium to be used for theprecultivation and the composition of the medium to be used for theproduction cultivation may be the same or different as required.

The cultivation is preferably conducted under aeration stirringconditions. For example, a known method such as stirring with apropeller or other machine, rotation or shaking of a fermenter,treatment with a pump, air blowing or the like is appropriatelyemployed. Sterile air is used for aeration.

The cultivation temperature can appropriately be changed within such arange that WF16616 substance-productive fungi produce WF16616 substance.It is usually between 9° and 39° C., preferably between 23° and 30° C.The cultivation time varies with the cultivation conditions or thecultivation amount. It is usually between approximately 1 day and 2weeks.

After the completion of the fermentation, the desired WF16616 substanceis recovered from the culture broth. That is, from the cultivated fungus(or the cultivated fungus-containing culture broth) the desiredsubstance is extracted directly with water and/or an organic solvent, orthe cultivated fungus is milled mechanically or by a known means such assupersonic waves or the like, then the desired substance being extractedwith water and/or an organic solvent, and recovered and purified in ausual manner. In the case of the filtered culture broth, it may bedirectly recovered and purified in a usual manner.

For recovering and purifying, usual methods may be employed whichinclude, for example, extraction with a solvent such as water, anorganic solvent or a mixed solvent thereof, chromatography andrecrystallization with a single solvent or a mixed solvent. Thesemethods may be adopted singly or in combination.

WF16616 substance is recovered and purified by the above-mentioned knownmethods. For example, the culture broth is subjected to extraction usingacetone, and the obtained extract is further subjected to ion-exchangeresin treatment and silica-gel treatment. The thus-obtained substance ispurified through chromatography. The purification is further repeated asrequired, and its dry powder can finally be obtained.

The physico-chemical properties of the thus-obtained WF16616 substanceare shown in Tables 3, 4 and 5.

Table 3

(1) Physico-chemical properties of a salt (considered to be a sodiumsalt, a potassium salt, an ammonium salt or a mixture thereof) ofWF16616 substance

(1) Color and state of the substance

white powder

(2) Molecular formula

C₅₁ H₈₂ N₈ O₂₁ S (as a free acid)

(3) Molecular weight

FAB-MS m/z 1291 (M+2Na-H)

(4) Decomposition point

210°-215° C. (decomp.)

(5) Specific rotation

α!_(D) ²³ : -20° (c=1.0; methanol)

(6) UV absorption spectrum

λ_(max) ^(H).sbsp.2^(O) : 270

λ_(max) ⁰.01N NaOH : 245, 290

Table 4

(2) Physico-chemical properties of WF16616 substance

(7) IR absorption spectrum

ν_(max) ^(KBr) : 3350, 2959, 2926, 2856, 1670, 1629, 1534, 1442, 1402,1273, 1148, 1048, 969, 946, 885, 805 cm⁻¹

(8) Solubility in a solvent

easily soluble in methanol

sparingly soluble in ethyl acetate and acetone

insoluble in chloroform

(9) Color reaction

positive: ninhydrin reaction, cerium sulfate reaction, iodine steamreaction

negative: ferric chloride reaction, Molish's reaction, Ehrlich'sreaction

(10) Classification of an acid substance and a basic substance

acid substance

Table 5

(3) Physico-chemical properties of WF16616 substance

(11) ¹ H nuclear magnetic resonance spectrum (500 MHz, CD₃ OD)

δ_(H) : 7.31(1H, d, J=2 Hz), 7.02(1H, dd, J=2 and 8 Hz), 6.86(1H, d J=8Hz), 5.24(1H, d, J=3 Hz), 5.06(1H, d, J=4 Hz), 4.94(1H, d J=3 Hz),4.59-4.48(3H, m), 4.47-4.34(5H, m), 4.28(1H, dd, J=2 and 6 Hz), 4.16(1H,m), 4.07(1H, m), 3.98-3.89(2H, m), 3.75(1H, d J=1 Hz), 3.35(1H, t J=10Hz), 2.76(1H, dd J=4 and 5 Hz), 2.51(1H, m), 2.45(1H, dd, J=8 and 16Hz), 2.39(1H, dd, J=7 and 16 Hz), 2.21(2H, t, J=7 Hz), 2.03-1.94(3H, m),1.57(2H, m), 1.50-1.38(2H, m), 1.37-1.20(12H, m), 1.17(3H d, J=6 Hz),1.12-1.04(3H, m), 1.05(3H, d, J=7 Hz), 0.90(1H, m), 0.87(3H, t, J=7 Hz),0.85(3H, d, J=6 Hz), 0.84(3H, d, J=6 Hz).

The chart is shown in FIG. 1.

(12) ¹³ C nuclear magnetic resonance spectrum (125 MHz, CD₃ OD)

δ_(c) : 176.9(s), 175.8(s), 174.4(s), 173.5(s), 172.7(s), 172.6(s),172.5(s), 169.3(s), 150.3(s), 141.1(s), 134.5(s), 125.6(d), 123.2(d),118.2(d), 76.4(d), 75.7(d), 75.5(d), 74.2(d), 71.3(d), 70.7(d), 70.6(d),70.1(d), 68.2(d), 62.4(d), 58.4(d), 57.1(t, CH₂), 56.9(d), 55.5(d),53.0(t, CH₂), 51.4(d), 45.9(t, CH₂), 39.6(t, CH₂), 39.1(d), 38.4(t,CH₂), 38.1(t, CH₂), 36.7(t, CH₂), 34.9(t, CH₂), 32.9(d), 31.2(d),31.1(t, CH₂), 30.7(t, CH₂), 30.6(t, CH₂), 30.3(t, CH₂), 30.3(t, CH₂),28.0(t, CH₂), 27.0(t, CH₂), 20.7(q), 20.2(q), 19.8(q), 11.6(q), ll.l(q).

The chart is shown in FIG. 2.

It is identified, as will be described later, that WF16616 substance ofthe present invention exhibits an excellent physiological activity,especially an excellent antifungal or antiprotozoan activity as well asa low toxicity and a high safety, and is quite effective as anantifungal agent or as an antiprotozoan agent.

Accordingly, the substance of the present invention can be used as anactive ingredient of a medication, and such a medication is alsoincluded in the present invention. The composition of the medication inthe present invention can be formed by adding an ordinary organic orinorganic carrier to WF16616 substance and/or its salt as an activeingredient. This composition is formulated into a solid, semi-solid orliquid oral administration agent or parenteral administration agent forexternal use, etc.

The oral administration agent may take the form of tablets, pills,granules, soft or hard capsules, powders, fine granules, grains,emulsions, suspensions, syrups, pellets or elixirs. The parenteraladministration agent may take the form of injections, drops, infusions,ointments, lotions, tonics, sprays, suspensions, oil solutions,emulsions and suppositories. The acitve ingredient of the presentinvention may be formulated into preparations in a usual manner. At thistime, ordinary adjuvant may be appropriately used as required. Examplesof the adjuvant include surfactants, excipients, coloring agents,flavors, preservatives, stabilizers, buffers, suspending agents andisotonic agents.

The dose of the composition in the present invention varies depending onthe type of the composition, the type of the disease to be prevented ortreated, the administration method, the age of the patient, the degreeof progression of the disease, the treatment time and the like. In thecase of intravenous administraiton, the dose of the active ingredient(WF16616 substance or/and its salt) is between 0.01 and 1,000 mg/kg,preferably between 0.1 and 100 mg/kg per day for an adult. In the caseof intramuscular administration, it is between 0.01 and 1,000 mg/kg,preferably between 0.1 and 100 mg/kg per day for an adult. In the caseof oral administration, it is between 0.5 and 2,000 mg/kg, preferablybetween 1 and 1,000 mg/kg per day for an adult.

The present invention is illustrated more specifically by referring tothe following Examples.

EXAMPLE 1

Fermentative Production of WF16616 Substance

(1) Incubation

An aqueous seed medium containing glycerol (2%), sucrose (2%),cottonseed meal (2%), dry yeast (1%), polypeptone (1%), KH₂ PO₄ (0.1%)and Tween 80 (0.1%) was added to each of three 500-ml Erlenmeyer flasksin an amount of 160 ml each, and was sterilized at 120° C. for 30minutes. One loopful of a slant culture of No. 16616 strain which hadbeen fully grown upon incubation in a YpSs agar medium at 25° C. for 2weeks was inoculated in the above-mentioned sterilized medium, and wasincubated at 25° C. for 1 week while being shaken using a rotary shakerat 220 rpm and a stroke of 5.1 cm.

An aqueous productive medium containing corn starch (2%), glucose (1%),cotton seed meal (1%), soybean meal (0.5%) and dry yeast (0.5%) wascharged into a 30-liter stainless steel jar fermenter, and wassterilized at 120° C. for 30 minutes.

The above-obtained seed culture was inoculated in 20 liters of thesterilized productive medium, and the fermentation was continued at 25°C. for 6 days while being stirred at 200 rpm upon aeration at a rate of20 liters/min.

(2) Separation and Purification

After the completion of the incubation, to 15 liters of the culturebroth, is added the same amount of acetone, and the mixture wassubjected to extraction at room temperature for 2 hours. To theresulting mixture, was added a diatomaceous earth for filtration. Theobtained filtrate was concentrated by removing acetone under reducedpressure. The pH of the concentrate was adjusted to 3.95 with conc. H₂SO₄, and the concentrate was then subjected to adsorption treatmentusing 0.75 liters of a high-molecular adsorbent Diaion HP-20 (made byMitsubishi Kagaku Co.). The column was washed with water and with 50%aqueous methanol, and elution was then carried out with methanol andwith acetone.

The eluate (3.75 liters) was evaporated to dryness under reducedpressure. The thus-obtained dry substnace was dissolved in a smallamount of methanol, and 100 ml of silica gel (Kiesel gel 60, made by E.Merck Co.) was added thereto to form a slurry. Methanol was removed, andthe residual dry powder was applied to a column (200 ml) of theabove-mentioned silica gel. The column was developed with ethyl acetate,with acetone and with a mixed solution of acetone and methanol.Two-hundred milliliters of the active fraction which was eluted with themixed solution of acetone and methanol (at a ratio of 10:1 and 1:1) wasconcentrated to dryness under reduced pressure.

The resulting residue was dissolved in 200 ml of 50% aqueous methanol,and was subjected to YMC-gel (ODS-AM, 120-S50, made by YMC, 350 ml)chromatography. The column was washed with 50% aqueous methanol and with60% aqueous methanol, and elution was carried out with 70% aqueousmethanol. Fractions containing the active substance were mixed togetherto come to 500 ml, and the 500 ml was concentrated under reducedpressure to obtain 160 ml of the resulting aqueous methanol solution.

This solution was subjected to preparative HPLC, YMC column (ODS-AM,SH-343-5AM, S-5, 250×20 mm i.d.) chromatography, and was eluted at aflow rate of 16 ml/min using 45% aqueous acetonitrile containing NH₄ H₂PO₄ (0.5%). One hundred milliliters of the active fraction wascollected, and diluted to twice with water. This solution was applied toa YMC column (ODS-AM, SH-343-5AM, S-5, 250×20 mm i.d.). The column waswashed with 50% aqueous methanol, and was then developed at theabove-mentioned flow rate using 60% aqueous methanol. One hundredmilliliters of the eluate was concentrated under reduced pressure, andwas then freeze-dried to obtain 46.6 mg of a white powder which is thesalt of WF16616 substance (considered to be a sodium salt, a potassiumsalt, an ammonium salt or a mixture thereof).

EXAMPLE 2

Biological Properties of WF16616 Substance

(1) Antifungal Activity

The antifungal activity of WF16616 substnace was measured in a 0.5%glucose-containing yeast nitrogen base (YNBD) medium by a microbrothdilution analysis method using a 96-well microtiter plate. Thissubstance was dissolved in methanol, and was diluted to twice insequence in the microtiter plate using the YNBD medium.

The test microorganism was inoculated at a concentration of 1×10⁴cfu/100 μl/well. Candida albicans and Aspergillus fumigatus wereincubated in the above-mentioned plate at 37° C. for 22 hours, andCryptococcus neoformans at 30° C. for 48 hours, respectively. Then, MICwas measured through microscopical observation. The results are shown inTable 6. As is clear from the results, it was identified that thesubstance of the present invention exhibited the outstanding antifungalactivity.

                  TABLE 6                                                         ______________________________________                                                          MIC (μg/ml)                                              ______________________________________                                        Candida albicans No. 7                                                                            0.78                                                      Candida albicans FP633                                                                            0.10                                                      Aspergillus fumigatus FD050                                                                       0.16                                                      Cryptococcus neoformans YC203                                                                     >50                                                       ______________________________________                                    

(2) Toxicity Test

WF16616 substance was intraperitoneally injected into each of five5-week-old ICR female mice at a dose of 5 mg/kg once a day for threedays. However, none of the mice were dead, and the increase in theweight of these mice was quite the same as that in the weight ofunadministered mice. It was thus identified that WF16616 substanceexhibited a high safety.

EXAMPLE 3

Production of an Injection

    ______________________________________                                        (1)     WF16616 substance produced in Example 1                                                                5 g                                          (2)     NaCl                     9 g                                          (3)     sodium hydrogencarbonate 1 g                                          ______________________________________                                    

All of the above-mentioned ingredients (1) to (3) were dissolved in 100ml of distilled water, and 1 ml of the resulting solution was chargedinto an ampoule. Thus, 100 ampoules of the injection were prepared.

Effects of the Invention

The present invention is to provide WF16616 substance. This substance isa novel substance which exhibits an excellent physiological activity andwhich can be used for various medications as antimicrobial agents,especially, e.g., as an antifungal agent and as an agent forPneumocystis carinii pneumonia.

Statement on the Microorganism Deposited Under Regulation No. 13-2

1. Tolypocladium parasiticum No. 16616

a. Name and address of a depository in which the above-mentionedmicroorganism was deposited: Name: National Institute of Bioscience andHuman-Technology of the Agency of Industrial Science and Technology,Ministry of International Trade and Industry Address: 1-3, Higashi 1chome, Tsukuba-shi, Ibaraki-ken, 305 Japan

b. Date on which the microorganism was deposited in the depositorydescribed in a: May 25, 1995

c. Deposit No. which was allotted by the depository described in a: FERMBP-5553

Additional Explanation on the Deposit of the Microorganism

The above-mentioned deposit, FERM BP-5553, is the deposit transferred onMay 30, 1996 from the National deposit No. FERM P-14941 deposited on May25, 1995 (the original deposit date.

We claim:
 1. WF16616 substance represented by the following structuralformula (1) or its pharmaceutically acceptable salt. ##STR3##
 2. Aprocess for producing WF16616 substance, which comprises culturing aWF16616 substance-productive fungus belonging to the genus Tolypocladiumto produce WF16616 substance, and isolating this substance.
 3. Theprocess of claim 2, wherein the WF16616 substance-productive fungus isTolypocladium parasiticum.
 4. A pharmaceutical preparation comprisingWF16616 substance or its pharmaceutically acceptable salt, and apharmaceutically acceptable, substantially non-toxic carrier orexcipient.
 5. The pharmaceutical preparation of claim 4 which is used asan antifungal agent.